Cloning, Sequencing and Cloning, Sequencing and expression in expression in Escherichia coliEscherichia coli of the Rubredoxin gene from of the Rubredoxin gene from Clostridium pasteurianumClostridium pasteurianum

Mathieu, I., Meyer, J., and Moulis, J. (1992)

J. Biochem. 285, (255-262)

Background: StructureBackground: Structure

• Non-heme proteins• Composed of 45 to

54 amino acid residues

• Majority occur in anaerobic bacterium

• Molecular weight ranging from 5000 and 6000 Daltons

Background: StructureBackground: Structure

Ribbon structure of Rubredoxin from Clostridium pasteurianum showing iron (orange core), and four Cystiene residues.

Background: FunctionBackground: Function

•Presumed to serve as electron carriers

•Electron-transfer chain in which they participate has only been identified in P. oleovorans

Purpose

Why study Rubredoxin:

• ETC is important to cellular function

• Structure is known, but not function

Goals:

• Develop a method for over-expression of

Rubredoxin

• Use resultant protein to study role in ETC

Cloning Step 1Cloning Step 1• Derived probes (p1) and (p2) for Rub• Digested Cpa genome with RE• Southern blotted using p1 and p2

Cloning Step 2Cloning Step 2

• Determined that Rub DNA appears at 3.9 kb by using gel electrophoresis

Cloning Step 3Cloning Step 3

• Digested Cpa with RE to isolate Rub sequence

• Sequence inserted into HindIII-BamHI pUC18

Cloning Step 4Cloning Step 4

• pUC18 transformed into E.coli DH5alpha cells

• Plated on amp plates

• Retested colonies by SB to ensure Rub gene transformed

• Rub gene was in fact transformed; One clone produced pCPRD1

SequencingSequencing

• pCPRD1 sequenced• BgLII-SspI no remarkable

features• ORF1: compared to known

reductases• ORF2: gene product has no

function• ORF3: compared to Cpa

reductases• ORF4: Rubredoxin gene• Specific site of Rub gene

found

Sequenced fragment taken from Cpa

Over-ExpressionOver-Expression

• Plasmid pCPRD 1 was moved to JM109 E.coli cells

• Added IPTG to increase expression

• Did not work

Over-ExpressionOver-Expression

• Made a second clone, pCPRD2, using specific sites identified on pCPRD1

• Plasmid pCPRD2 was moved to JM109 E.coli cells

• Added IPTG

• Used UV spectroscopy to identify time at which IPTG was most effective:

• After 1hr detectable expression

• After 4hr leveled off

• Stable for at least 24 hrs

• At optimum time, proteins were harvested

DiscussionDiscussion

• Determined that Rubredoxin is generated in one piece• Rub function in Cpa still unknown• Found no direction connection between ORF1/ORF3

and Rub• Even though E.coli does not contain naturally

occurring Rub, it is efficient in expressing foreign proteins that have elaborate iron-sulfur clusters

• Method using Cpa in E.coli and IPTG produces substantially more protein than Cpa

DiscussionDiscussion

• Amino acid sequence of cloned Rub gene compared to known sequences of Rub and found to have conserved residues.

DiscussionDiscussion

• Compared UV spec of Rub protein (naturally occurring) to Rub protein (in E.coli) and found them to be the same.

Questions?

What is ETC?